(1) Field of the Invention
This invention relates generally to trophic or growth factors and, more particularly, to novel growth factors of the neurturin-GDNF family of growth factors.
(2) Description of the Related Art
The development and maintenance of tissues in complex organisms requires precise control over the processes of cell proliferation, differentiation, survival and function. A major mechanism whereby these processes are controlled is through the actions of polypeptides known as xe2x80x9cgrowth factorsxe2x80x9d. These structurally diverse molecules act through specific cell surface receptors to produce these actions.
Growth factors, termed xe2x80x9cneurotrophic factorsxe2x80x9d promote the differentiation, growth and survival of neurons and reside in the nervous system or in innervated tissues. Nerve growth factor (NGF) was the first neurotrophic factor to be identified and characterized (Levi-Montalcini et al., J. Exp. Zool. 116:321, 1951 which is incorporated by reference). NGF exists as a non-covalently bound homodimer that promotes the survival and growth of sympathetic, neural crest-derived sensory, and basal forebrain cholinergic neurons. In sympathetic neurons this substance produces neurite outgrowth in vitro and increased axonal and dendritic in growth in vivo. (See Levi-Montalcini and Booker, Proc. Nat""l Acad Sci 46:384-391, 1960; Johnson et al. Science 210:916-918, 1980; Crowley et al., Cell 76:1001-12, 1994 which are incorporated by reference). NGF has effects on cognition and neuronal plasticity, and can promote the survival of neurons that have suffered damage due to a variety of mechanical, chemical, viral, and immunological insults (Snider and Johnson, Ann Neurol 26:489-506, 1989; Hefti, J. Neurobiol 25:1418-35, 1994 which are incorporated by reference). NGF also is known to extensively interact with the endocrine system and in immune and inflammatory processes. (Reviewed in Scully and Otten, Cell Biol Int 19:459-469, 1995; Otten and Gadient, Int. J. Devl Neurosci 13:147-151, 1995 which are incorporated by reference). For example, NGF promotes the survival of most cells. (Horigome et al. J Biol Chem 269:2695-2707, 1994 which is incorporated by reference).
In recent years it has become apparent that growth factors fall into classes, i.e. families or superfamilies based upon the similarities in their amino acid sequences. These families include, for example, the fibroblast growth factor family, the neurotrophin family and the transforming growth factor-beta (TGF-xcex2) family. As an example of family member sequence similarities, TGF-xcex2 family members have 7 canonical framework cysteine residues which identify members of this superfamily.
NGF is the prototype of such a family of growth factors. Brain-derived neurotrophic factor (BDNF), the second member of this family to be discovered, was shown to be related to NGF by virtue of the conservation of all six cysteines that form the three internal disulfides of the NGF monomer (Barde, Prog Growth Factor Res 2:237-248, 1990 and Liebrock et al. Nature 341:149-152, 1989 which are incorporated by reference). By utilizing the information provided by BDNF of the highly conserved portions of two factors, additional members (NT-3, NT-4/5) of this neurotrophin family were rapidly found by several groups (Klein, FASEB J 8:738-44, 1994 which is incorporated by reference).
Neurotrophic factors structurally unrelated to NGF have been recently identified. These include factors originally isolated based upon a xe2x80x9cneurotrophic actionxe2x80x9d such as ciliary neurotrophic factor (CNTF) (Lin et al., Science 246:1023-5, 1989 which is incorporated by reference) along with others originally isolated as a result of non-neuronal activities (e.g. fibroblast growth factors (Cheng and Mattson Neuron 1:1031-41, 1991 which is incorporated by reference), IGF-I (Kanje et al, Brain Res 486:396-398, 1989 which is incorporated by reference) leukemia inhibitory factor (Kotzbauer et al, Neuron 12:763-773, 1994 which is incorporated by reference).
Glial-derived neurotrophic factor (GDNF), is one such neurotrophic factor structurally unrelated to NGF. GDNF was, thus, a unique factor, which, up until now, was not known to be a member of any subfamily of factors. The discovery, purification and cloning of GDNF resulted from a search for factors crucial to the survival of midbrain dopaminergic neurons, which degenerate in Parkinson""s disease. GDNF was purified from rat B49 glial cell conditioned media (Lin et al., Science 260:1130-2, 1993 which is incorporated by reference). Sequence analysis revealed it to be a distant member of the TGF-xcex2 superfamily of growth factors, having approximately 20% identity based primarily on the characteristic alignment of the 7 canonical framework cysteine residues (Lin et al., Science 260:1130-2, 1993 which is incorporated by reference). Thus, GDNF could possibly have represented a new subfamily within the TGF-xcex2 superfamily.
Recombinant GDNF produced in bacteria specifically promotes the survival and morphological differentiation of dopaminergic neurons (Lin et al., Science 260:1130-2, 1993); Tomac et al., Nature 373:335-9, 1995; Beck et al., Nature 373:339-41, 1995 and Ebendal et al., J Neurosci Res 40:276-84, 1995 which are incorporated by reference) and motor neurons (Henderson et al., Science 266:1062-4, 1994; Yan et al., Nature 373:341-4, 1995; and Oppenheim et al., Nature 373:344-6, 1995 which are incorporated by reference). Overall, GDNF was a more potent factor for promoting the survival of motor neurons than the other factors, and it was the only factor that prevented neuronal atrophy in response to these lesions, thereby positioning it as a promising therapeutic agent for motor neuron diseases.
It is now generally believed that neurotrophic factors regulate many aspects of neuronal function, including survival and development in fetal life, and structural integrity and plasticity in adulthood. Since both acute nervous system injuries as well as chronic neurodegenerative diseases are characterized by structural damage and, possibly, by disease-induced apoptosis, it is likely that neurotrophic factors play some role in these afflictions. Indeed, a considerable body of evidence suggests that neurotrophic factors may be valuable therapeutic agents for treatment of these neurodegenerative conditions, which are perhaps the most socially and economically destructive diseases now afflicting our society. Nevertheless, because different neurotrophic factors can potentially act preferentially through different receptors and on different neuronal or non-neuronal cell types, there remains a continuing need for the identification of new members of neurotrophic factor families for use in the diagnosis and treatment of a variety of acute and chronic diseases of the nervous system.
Briefly, therefore, the present invention is directed to the identification and isolation of substantially purified factors that promote the survival and growth of neurons as well as non-neuronal cells. Accordingly, the inventors herein have succeeded in discovering novel protein growth factors belonging to a family of growth factors for which GDNF was the first known member. The first such newly discovered family member was neurturin and this is the subject of application Ser. No. 08/519,777, now U.S. Pat. No. 5,739,307. Based upon the sequence of GDNF and neurturin the inventors herein have discovered another member of the GDNF-Neurturin family of growth factors referenced herein as persephin (PSP). This growth factor is believed to show at least 85% sequence identity among homologous sequences from different mammalian species although sequence homology may be as low as 65% in non-mammalian species such as avian species. Persephin proteins identified herein include mouse sequences as set forth in SEQ ID NOS:79, 80 and 81 (FIG. 11; amino acid residues 52 through 140, 47 through 142, and 9 through 142, respectively) and rat sequences as set forth in SEQ ID NOS:82 and 83 (FIG. 14; amino acid residues 1 through 89 and 1 through 91, respectively). In addition, human persephin is identified by virtue of its having at least 85% sequence homology with its ortholog, mature mouse persephin, along with the identification of certain conserved amino acid residues contained within human persephin as shown in FIG. 15. Thus, it is believed that human persephin will have 28 amino acids in the aligned sequence between the first and seventh canonical framework cysteine residues as set forth in FIG. 15 with residues numbered from the N-terminal end of the family member aligned sequence being (1) Cys, (3) Leu, (10) Val, (13) Leu, (14) Gly, (15) Leu, (16) Gly, (17) Tyr, (21) Glu, (25) Phe, (26) Arg, (27) Tyr, (28) Cys, (30) Gly, (32) Cys, (44) Leu, (47) Leu, (58) Cys, (59) Cys (61) Pro, (66) Asp, (69) Phe, (70) Leu, (71) Asp, (83) Ser, (84) Ala, (87) Cys, and (89) Cys.
Persephin has been identified and obtained by a method based upon the conserved regions of the GDNF-Neurturin family discovered by the inventors herein. Accordingly, a new method has been devised that utilizes degenerate primers constructed from the sequences of these conserved regions for use in the polymerase chain reaction procedure. By utilizing this method the mouse and rat orthologs of the new family member, persephin, have been identified and obtained.
The present invention thus provides both amino acid sequences and nucleotide sequences that encode mouse and rat persephin as set forth in the amino acid sequences of SEQ ID NO:79-83 and nucleotide sequences of SEQ ID NOS:84 and 85. Because of the close homology between the mouse and rat sequences (95% sequence identity), it is believed that the human persephin sequence will show a high sequence homology to the mouse and rat sequences.
Expression vectors and stably transformed cells are also within the scope of this invention. The transformed cells can be used in a method for producing persephin.
In another embodiment, the present invention provides a method for preventing or treating neuronal degeneration comprising administering to a patient in need thereof a therapeutically effective amount of persephin. A patient may also be treated by implanting transformed cells which express persephin or a DNA sequence which encodes persephin into a patient, or cells cultured and expanded by growth in persephin.
The present invention also provides compositions and methods for detecting persephin. One method is based upon persephin antibodies and other methods are based upon detecting mRNA and cDNA or genomic DNA encoding persephin using recombinant DNA techniques.
Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of a new growth factor, persephin, for use in preventing the atrophy, degeneration or death of certain cells, in particular neurons; the provision of human persephin by making available the specific sequences of murine and rat persephin from which the human sequence can be identified and obtained; the provision of other members of the neurturin-persephin-GDNF family of growth factors by making available new methods capable of obtaining other family members; the provision of methods for obtaining persephin by recombinant techniques; the provision of methods for preventing or treating diseases producing cellular degeneration and, particularly neuronal degeneration; the provision of methods that can detect and monitor persephin levels in a patient; and the provision of methods that can detect alterations in the persephin gene.